Anti spi1

The RIP assay was performed via the EZ-Magna RIP RNA-binding Protein Immunoprecipitation Kit (Millipore, Darmstadt, Germany) as previously described [8 (link)]. Briefly, GSCs were lysed in RIP buffer, incubated with magnetic beads conjugated with anti-SPI1, anti-DGCR8 antibodies or negative control IgG (Abcam). Then the protein-RNAs complex was immunoprecipitated, and RNAs were isolated with proteinase K. Finally, RNAs were purified, and qPCR was used to check the circKPNB1 expression in the precipitants.

Protein levels were analyzed using IB and ELISA following conventional protocols. For IB, the primary antibodies used were anti-YAP (Abcam, Cambridge, MA, USA, #ab52771), anti-GAPDH (CST, Boston, MA, USA, #5174 and #51332), anti-p-127-YAP (Abcam, #ab76252), anti-p-381-YAP (CST, #13619), anti-O-Glc-YAP (developed by Biolynx, Hangzhou, China), anti-HRS (Abcam, #ab72053), anti-PLD2 (CST, #13904), anti-RAB2B (Novus, Littleton, CO, USA, #NBP1-31631), anti-RAB27A (Abcam, #ab55667), anti-RAB27B (Abcam, #ab103418), anti-VAMP (Abcam, #ab36195), anti-ATG7 (Abcam, #ab52472), anti-SOX10 (Abcam, #ab227680), anti-Mycn (Abcam, #ab227822), anti-ALYREF (Abcam, #ab202894), anti-NSUN2 (Abcam, #ab259941), anti-DNMT2 (Abcam, #ab220175), anti-NSUN1 (Abcam, #ab270175), anti-NSUN3 (Abcam, #ab272616), anti-NSUN4 (Abcam, #ab235430), anti-NSUN5 (Abcam, #ab121633), anti-NSUN6 (Novus, #NBP1-92202), anti-NSUN7 (Biorbyt, Cambridge, UK, #orb258175), anti-YBX1 (Abcam, #ab255606), anti-EXOSC10 (Abcam, #ab94981), anti-SPI1 (Abcam, #ab227835), anti-CD63 (Abcam, #ab271286), anti-TSG101 (Abcam, #12501), anti-ALIX (Abcam, #ab275377), anti-CD9 (Abcam, #ab92726), and anti-calnexin (Abcam, #ab133615). For ELISA, YAP and NSUN2 levels were measured using kits from Yingxin Biotech Ltd. (Shanghai, China) as per manufacturer’s instructions.